(精选)【医学课件课件大全】 蛋白质电泳教学课件.ppt

蛋白质电泳;原 理 ;电泳原理示意图;在蛋白质组学中对电泳的分类;一维电泳;凝胶浓度和蛋白质分离范围;缓冲液的选择;12%胶常用的低分子量标准蛋白;双向电泳(2DE);2DE仪器系统;2DE在蛋白质组学方案中所处的位置;双向电泳的操作程序(以进行差别表达蛋白组分析为例);目前，普遍采用预制的固定pH梯度胶条(immobilized pH gradient strips, IPG strips)。商品化的IPG strips可以从Amersham Pharmacia公司或BioRad公司购买。;IPG胶条制备(casting of IPG strips);;Procedure; Assembly of the gel casting cassette ;IPG gel casting ;The acidic, dense solution is pipetted into the mixing chamber and the basic, light solution into the reservoir of the gradient mixer, an extra portion of the dense solution is prepared and pipetted into the mold prior to pouring the gradient. ;After pouring the gradient into the precooled mold (refrigerator), the mold is kept at room temperature for 15 min to allow adequate levelling of the density gradient prior to polymerization for one hour at 50°C. After polymerization, the mold is kept at room temperature for at least 15 min. Then the IPG gel is removed from the mold and extensively washed with deionized water, impregnated with 2% glycerol, and dried at room temperature in a dust-free cabinet and, if not used immediately, covered with a plastic film for storage at -20°C. The dried gels can be stored frozen for at least one year. ;Note: In order to ensure the reproducibility of the IPG gradient, the volume of the cassette should be constant. Therefore, it is recommended to check the volume of the cassette from time to time since it diminishes on ageing of the U-frame. ;Note: When one of the chambers is emptying faster than the other, the resulting pH gradient will not be linear. Check if there is an air bubble in the connecting line or whether the speed of the magnetic stirrer is not appropriate! ;1. 样品制备;对于脂蛋白，膜蛋白的分析，可以用如下的蛋白质裂解液： 7M urea, 2M thiourea, 4% CHAPS, 40 mM Tris base, 65 mMDTE;Presulubilization of protein in (boiling) SDS buffer, followed by dilution with urea lysis buffer. Initially solubilized in 0.5-1% SDS,followed by dilution with at least an eight excess of 2-4% (w/v) NP40, Triton X-100, or CHAPS to